CD84 is a Suppressor of T and B Cell Activation during Mycobacterium tuberculosis Pathogenesis

Abstract

Interest in host-directed therapies as alternatives/adjuncts to antibiotic treatment has resurged with the increasing prevalence of antibiotic-resistant tuberculosis (TB). Immunotherapies that reinvigorate immune responses by targeting immune checkpoints like PD-1/PD-L1 have proved successful in cancer therapy. Immune cell inhibitory receptors that trigger Mycobacterium tuberculosis-specific immunosuppression, however, are unknown. Here, we show that the levels of CD84, a SLAM family receptor, increase in T and B cells in lung tissues from M. tuberculosis-infected C57BL/6 mice and in peripheral blood mononuclear cells (PBMCs) from pulmonary TB patients. M. tuberculosis challenge experiments using CD84-deficient C57BL/6 mice suggest that CD84 expression likely leads to T and B cell immunosuppression during M. tuberculosis pathogenesis and also plays an inhibitory role in B cell activation. Importantly, CD84-deficient mice showed improved M. tuberculosis clearance and longer survival than M. tuberculosis-infected wild-type (WT) mice. That CD84 is a putative M. tuberculosis infection-specific inhibitory receptor suggests it may be a suitable target for the development of TB-specific checkpoint immunotherapies. IMPORTANCE Immune checkpoint therapies, such as targeting checkpoints like PD-1/PD-L1, have proved successful in cancer therapy and can reinvigorate immune responses. The potential of this approach for treating chronic infectious diseases like TB has been recognized, but a lack of suitable immunotherapeutic targets, i.e., immune cell inhibitory receptors that trigger immunosuppression specifically during Mycobacterium tuberculosis pathogenesis, has limited the application of this strategy in the development of new TB therapies. Our focus in this study was to address this gap and search for an M. tuberculosis-specific checkpoint target. Our results suggest that CD84 is a putative inhibitory receptor that may be a suitable target for the development of TB-specific checkpoint immunotherapies.

Keywords: CD84; Mycobacterium tuberculosis; checkpoint immunotherapy; pathogenesis.

FIG 1

SLAM family member CD84 is more highly expressed in the lungs of M. tuberculosis H37Rv-infected C57BL/6 mice than in the lungs of uninfected mice and in PBMCs from pulmonary TB patients than in PBMCs from healthy donors. (a to e) Total RNA was extracted from lung tissue from M. tuberculosis H37Rv-infected (1 × 10⁶ CFU) and uninfected C57BL/6 mice (n = 3) 30 days postinfection and was sequenced on an Illumina HiSeq X Ten system (three independent biological replicates). (a) Scatterplot of the expression of all genes. (b) Heat map of genes specifically upregulated (1,499) and downregulated (361) in the lung transcriptome of mice infected with M. tuberculosis. (c) GO classifications of differentially expressed genes. (d) KEGG enrichment of differentially expressed genes. (e) RNA-Seq data (reads per kilobase per million [RPKM] values) for the expression of SLAMF family proteins in lung tissue from M. tuberculosis-infected and uninfected mice. (f) qPCR analysis of SLAMF1, Ly9, and CD84 expression in lung tissue from M. tuberculosis-infected and uninfected mice, 30 days postinfection. (g) Flow cytometry of lung lymphocytes from M. tuberculosis-infected and uninfected mice stained by PE–anti-CD84 antibody; histogram showing the percentages of lung lymphocytes expressing CD84. (h) Flow cytometry of PBMCs derived from patients with pulmonary TB and healthy donors and stained with PE–anti-CD84 antibody; histogram showing the percentages of PBMCs expressing CD84. Data presented are mean values ± SD from three independent experiments. ***, P < 0.001 (Student’s t test).

FIG 2

CD84 is more highly expressed on T cells from M. tuberculosis-infected than on T cells from uninfected mice and on PBMCs from pulmonary TB patients than on PBMCs from healthy donors. (a, b) Total RNA was extracted from T cells separated from the lung and spleen tissues of M. tuberculosis-infected (1 × 10⁶ CFU H37Rv) and uninfected C57BL/6 mice 30 days and 60 days postinfection. (a) Levels of CD84 mRNA in CD4⁺ and CD8⁺ T cells from the lung and spleen of M. tuberculosis-infected and uninfected mice, as determined by qPCR. (b) Histogram showing the percentages of CD4⁺ and CD8⁺ T cells expressing CD84 in lung and spleen tissues from M. tuberculosis-infected and uninfected mice, as determined by flow cytometry. (c) Flow cytometry of PBMCs from patients with pulmonary TB and healthy donors, stained by PerCP–anti-CD4 antibody, APC–anti-CD8 antibody, and PE–anti-CD84 antibody. Data presented are mean values ± SD from three independent experiments; n = 3 for each group in each experiment. **, P < 0.01; ***, P < 0.001 (Student’s t test).

FIG 3

CD84 deficiency activates T cell immune responses during M. tuberculosis pathogenesis. (a, b) T cells were isolated from lung and spleen tissues from M. tuberculosis-infected (1 × 10⁶ CFU strain H37Rv) and uninfected WT and CD84-deficient C57BL/6 mice 30 and 60 days postinfection. (a) Percentages of CD4⁺ and CD8⁺ T cells in lung and spleen tissues from M. tuberculosis-infected WT or CD84-deficient mice 60 days postinfection, as determined by flow cytometry. (b) Percentages of CD69⁺ CD4⁺ and CD69⁺ CD8⁺ T cells in lung and spleen tissues from M. tuberculosis-infected WT and CD84-deficient mice 60 days postinfection, as determined by flow cytometry. (c) Percentages of CD84⁺ or CD84⁻ CD4⁺ and CD8⁺ T cells from pulmonary TB patients and healthy donors, as determined by flow cytometry after staining with PerCP–anti-CD4 antibody, APC–anti-CD8 antibody, and FITC–anti-CD69 antibody. (d) Cytokine levels in serum from M. tuberculosis-infected and uninfected WT and CD84-deficient mice 60 days and 80 days postinfection. Quantibody multiplexed quantitative sandwich ELISA array data for 12 TB-related cytokines. (e) Validation of IFN-γ expression at 80 days postinfection using standard ELISAs. (f) IFN-γ release from CD84⁺ and CD84⁻ PBMCs from pulmonary TB patients and healthy donors as determined using ELISA. CD84⁺ and CD84⁻ T cells were activated with soluble anti-CD3 antibody (clone OKT3) and ESAT-6 (5 μg/mL). (g) IFN-γ expression in differentiated Th1 cells as determined by flow cytometry. Sorted naive CD4⁺ T cells derived from PBMCs were cultured for 72 h under Th1-differentiating conditions and then stimulated for 5 h with 2 μL/mL cell activation cocktail. Data correspond to mean values ± SD from three independent experiments; n = 3 for each group in each experiment. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, nonsignificant (Student’s t test).

FIG 4

CD84 inhibits B cell activation and antibody production in response to M. tuberculosis infection in mice. (a to c) RNA-Seq of total RNA from lung tissues from M. tuberculosis-infected (1 × 10⁶ CFU strain H37Rv) WT and CD84-deficient C57BL/6 mice 30 days postinfection. (a) Scatterplot of the expression of all genes. (b) KEGG enrichment of differentially expressed genes. (c) qPCR determination of CD84 mRNA levels in B cells (left) and percentages of CD84⁺ B cells in spleen tissues from M. tuberculosis-infected and uninfected WT and CD84-deficient mice 30 days postinfection (right), as determined by flow cytometry. (d) CD69 expression on B cells in spleen tissue from M. tuberculosis-infected and uninfected WT and CD84-deficient mice 60 days postinfection, as determined by flow cytometry. (e) Antibody levels in sera from M. tuberculosis-infected and uninfected WT and CD84-deficient mice 60 days and 80 days postinfection, as determined using Quantibody multiplexed quantitative sandwich ELISA arrays. (f) Immunohistology of spleen tissue from M. tuberculosis-infected (1 × 10⁶ CFU H37Rv) WT and CD84-deficient C57BL/6 mice 60 days postinfection, stained with GC B cell markers PE-IgD (red) and FITC-GL7 (green). (g, h) Average GC size (g) and percentage of area occupied by GCs (h) as quantified using a Zeiss Axioplan microscope morphometric tool. Data presented are mean values ± SD from three independent experiments; n = 3 for each group in each experiment. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, nonsignificant (Student’s t test).

FIG 5

CD84 deficiency may promote M. tuberculosis clearance and survival. (a) Lung and spleen necroscopy of M. tuberculosis-infected (∼1 × 10⁶ CFU strain H37Rv) CD84-deficient and WT C57BL/6 mice 60 days postinfection. (b) Lung bacterial loads for M. tuberculosis-infected CD84-deficient and WT mice at the time points indicated (postinfection). (c) Left, H&E staining (×10 magnification, scale bar = 200 μm) and inset images of AF-stained lung tissue samples (×100 magnification, scale bar = 20 μm) for M. tuberculosis-infected CD84-deficient and WT mice showing lung pathology and the presence of M. tuberculosis bacilli in lung tissues 60 days postinfection. Right, histological score (area occupied by inflammation as a percentage of the total surface area in H&E-stained lung tissue sections). (d) Survival curve for a chronic infection model in which WT and CD84-deficient mice (n = 12) were infected with ∼1 × 10⁸ CFU M. tuberculosis H37Rv. Data presented in panels b and c represent mean values ± SD from representative experiments; n = 6 mice per group, three independent biological replicates being used in each case. ***, P < 0.001; ns, nonsignificant (Student’s t test).